Inhibition of reverse transcriptase activity increases stability of the HIV-1 core - PubMed (original) (raw)

Inhibition of reverse transcriptase activity increases stability of the HIV-1 core

Yang Yang et al. J Virol. 2013 Jan.

Abstract

Previous studies showed that HIV-1 reverse transcription occurs during or before uncoating, linking mechanistically reverse transcription with uncoating. Here we show that inhibition of reverse transcriptase (RT) during HIV-1 infection by pharmacologic or genetic means increased the stability of the HIV-1 core during infection. Interestingly, HIV-1 particles with increased core stability were resistant to the core-destabilizing effects of rhesus TRIM5α (TRIM5α(rh)). Collectively, this work implies that the surface of the HIV-1 core is dynamic and changes upon the ongoing processes within the core.

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Figures

Fig 1

Fig 1

Effects of reverse transcriptase inhibitors on HIV-1 core stability. (A) Cf2Th cells transduced with the empty vector LPCX were challenged with increasing amounts of HIV-1 GFP-reporter virus (800 pg/ml of p24) in the presence of AZT. As a control, LPCX-transduced cells were challenged in the presence of DMSO, the solvent used to resuspend the RT inhibitors. GFP-positive cells were quantified by flow cytometry. (B) LPCX-transduced Cf2Th cells in the presence of AZT or DMSO were challenged with similar amounts of HIV-1 GFP-reporter, and the amount of soluble versus particulate capsid was determined by the FOC assay. As control, we used the same amount of HIV-1 GFP-reporter to perform the FOC assay in Cf2Th cells stably expressing TRIM5αrh, which is expressed from the LPCX vector. Briefly, cells were incubated with HIV-1 GFP-reporter at 4°C for 30 min, washed, and returned to 37°C. Infection was allowed to proceed for 16 h. Cell extracts were fractionated on a sucrose cushion. Input, soluble, and pellet fractions were analyzed by Western blotting using antibodies against the HIV-1 p24 capsid protein. (C) The percentage of pelletable HIV-1 capsid was determined with respect to the amount of total input capsid. Similar results were obtained in three independent experiments, and standard deviations are shown. Statistical differences are given as P < 0.001 (two-way analysis of variance [ANOVA] followed by the Bonferroni posttest). (D) Cf2Th cells stably transduced with TRIM5αrh and selected in puromycin were analyzed for TRIM5αrh-hemagglutinin (HA) expression by Western blotting using anti-HA antibodies. The loading control was performed using anti-β-actin antibodies.

Fig 2

Fig 2

Effects of RT mutants on stability of the HIV-1 core. (A) Cf2Th cells transduced with the empty vector LPCX were challenged with the indicated HIV-1-GFP reporter viruses normalized by ELISA against p24 (800 pg/ml of p24). To study the effect of reverse transcription on core stability, we challenged cells with an HIV-1 strain bearing a mutation in the active site of the RT enzyme (HIV-1 RT D185N). Forty-eight hours postinfection, GFP-positive cells were quantified by flow cytometry. (B) Because the HIV-1 RT D185N mutant is not infectious, Gag-processing levels in viral supernatants were evaluated by Western blotting using antibodies against p24. (C) LPCX-transduced Cf2Th cells were challenged with similar amounts of the indicated HIV-1 GFP-reporter viruses, and the amount of soluble versus particulate capsid was determined by FOC assay. As a control, we used the same amount of wild-type HIV-1 GFP-reporter virus to perform the FOC assay in Cf2Th cells stably expressing TRIM5αrh, which is expressed from the LPCX vector. Briefly, cells were incubated with the indicated viruses at 4°C for 30 min, washed, and returned to 37°C. Infection was allowed to proceed for 16 h. Cell extracts were fractionated on a sucrose cushion. Input, soluble, and pellet fractions were analyzed by Western blotting using antibodies against the HIV-1 p24 capsid protein. (D) The percentage of pelletable HIV-1 capsid was determined with respect to the amount of total input capsid. Similar results were obtained in three independent experiments, and standard deviations are shown. Statistical differences are given as P < 0.01 (two-way ANOVA followed by the Bonferroni posttest).

Fig 3

Fig 3

The core-stabilizing HIV-1 RT mutant is resistant to the destabilizing effects of TRIM5αrh. (A) Cf2Th cells transduced with the empty vector LPCX or expressing TRIM5αrh were challenged with the indicated HIV-1 GFP-reporter viruses normalized by ELISA against p24. The amount of soluble versus particulate capsid was determined by FOC assay. Briefly, cells were incubated with the indicated viruses at 4°C for 30 min, washed, and returned to 37°C. Infection was allowed to proceed for 16 h. Cell extracts were fractionated on a sucrose cushion. Input, soluble, and pellet fractions were analyzed by Western blotting using antibodies against HIV-1 p24 capsid protein. (B) The percentage of pelletable HIV-1 capsid was determined with respect to input capsid. Similar results were obtained in three independent experiments, and standard deviations are shown. Statistical differences are given as P < 0.001 (two-way ANOVA followed by the Bonferroni posttest).

Fig 4

Fig 4

The use of AZT and nevirapine prevents the core-destabilizing effects of TRIM5αrh. (A) Cf2Th cells stably transduced with TRIM5αrh were challenged with the indicated HIV-1 GFP-reporter viruses, normalized by ELISA against p24, in the presence of AZT and nevirapine. The amount of soluble versus particulate capsid was determined by FOC assay. Briefly, cells were incubated with the indicated viruses at 4°C for 30 min, washed, and returned to 37°C. Infection was allowed to proceed for 16 h. Cell extracts were fractionated on a sucrose cushion. Input, soluble, and pellet fractions were analyzed by Western blotting using antibodies against HIV-1 p24 capsid protein. (B) The percentage of pelletable HIV-1 capsid was determined with respect to input capsid. Similar results were obtained in three independent experiments, and standard deviations are shown. Statistical differences are given as P < 0.05 (two-way ANOVA followed by the Bonferroni posttest).

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